1. Field of the Invention
The present invention is related generally to decontamination procedures and more specifically to decontamination procedures carried out using vapor phase hydrogen peroxide.
Contamination by microorganisms is one of the most troublesome problems encountered today by people growing tissue cultures. Contamination wastes time and money, and causes the loss of valuable tissue cultures. In addition to contamination problems, plant tissue culturists face problems stemming from the practice of "disinfesting" plant cells prior to culturing. Currently, there is no convenient method for disinfesting plant tissue without risking damage to the plant tissue itself.
No known methods exist for the decontamination of tissue culture incubators with the cultures in place, i.e. in situ decontamination, because known decontamination procedures, e.g. steam or ethylene oxide treatments, would destroy the cultures. Because of this lack of an ability to perform in situ decontamination, various technics have been employed to minimize the chances of tissue culture contamination. Incubators have been designed to provide vertical laminar air flow over the culture containers. This laminar air flow helps to keep airborne contaminants from coming into contact with the tissue cultures. One manufacturer has developed an incubator with copper walls which possesses germicidal and fungicidal properties under the proper conditions. Such an incubator cannot, however, destroy contaminants in the air, on the shelving, or on the various tissue culture containers. In the past, antibiotics have been added to tissue cultures to prevent contamination. However, genetic or metabolic changes can occur in the cells and it is now desirable to avoid adding antibiotics to the growth medium when at all possible. Thus, a need exists for a method which will enable the decontamination of tissue culture incubators with the cultures in place.
Plant cell culturists are currently required to use crude methods to disinfest plant cells prior to culturing experiments. Current practices involve soaking samples in dilute Chlorox (1%), glutaraldehyde, or ethanol for up to thirty minutes. Many plant cells cannot be exposed to these disinfectants. However, no real alternatives exist. Trial and error is the general rule employed to obtain pure cultures. Thus the need exists for a method which will enable plant cell culturists to disinfest plant cells prior to culturing experiments.
It is known that even low concentrations of vapor phase hydrogen peroxide can be effective for decontamination and sterilization. Although vapor phase hydrogen peroxide has been used in the past for such purposes, such uses have not involved living cells. For example, see Coulter U.S. Pat. No. 2,193,622 entitled "Preserving Bakery Products", Moore, et al. U.S. Pat. No. 4,169,123 entitled "Hydrogen Peroxide Vapor Sterilization Method", and Forstrom, et al. U.S. Pat. No. 4,169,124 entitled "Cold Gas Sterilization Process". In each of these patents, vapor phase hydrogen peroxide is used for sterilization but not in an environment containing living tissue.
Liquid phase hydrogen peroxide has been used around immobilized whole cells for the purpose of providing increased oxygenation, not for the purpose of decontamination. Holst, "Hydrogen Peroxide as an Oxygen Source for Immobilized Gluconobacter oxydans", Applied Microbiology and Biotechnology 22, pages 383-388. The major disadvantage of this process is that the hydrogen peroxide concentration in the liquid phase (34 mg/l) destroyed a majority of the cells prior to enzymatic decomposition of the hydrogen peroxide into oxygen and water.
Clearly, the need exists for a convenient method of in situ decontamination and a method for disinfesting plant cells prior to being cultured.